Difference between revisions of "Notes"
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| − | + | Snakes 1,2,3 not fed | |
| + | Snakes 4,5,6 fed | ||
| + | --info in excel file | ||
| + | RNA seq in multiplex --12 different samples, amplified with unique barcode (3 letter code) - simplified in excel file as individual letters. | ||
| + | |||
| + | '''How to seq RNA:''' | ||
| + | -snakes fed and decapitated upon mouse tail disappearing | ||
| + | -organs removed and flash frozen to preserve RNA | ||
| + | -tissue from organ shaved off to ~.1g to begin RNA isolation kit (2 organs, 6 snakes, 12 total samples) | ||
| + | ----may not have sampled right part of organ, may have sampled connective tissue rather than proper organ | ||
| + | -use mRNA to make cDNA copy, cDNA more stable | ||
| + | ---used magnetic beads with TTTTTT (poly T) end that binds to AAAAA (poly A), use magnet to attract beads, attached are mRNA | ||
| + | ---High throughput sequencing gives only short reads (~75 base pairs from each end) | ||
| + | -fragment mRNA into smaller strands so that with short reads ^^ each bp can be read | ||
| + | -use reverse transcriptase (RT)- enzyme in HIV/retroviruses- to form cDNA from mRNA + nucleotides | ||
| + | -primers made of every possible 6 letter bp combination --> allows us to amplify every primer fragment | ||
Revision as of 19:55, 12 January 2016
Snakes 1,2,3 not fed Snakes 4,5,6 fed --info in excel file RNA seq in multiplex --12 different samples, amplified with unique barcode (3 letter code) - simplified in excel file as individual letters.
How to seq RNA: -snakes fed and decapitated upon mouse tail disappearing -organs removed and flash frozen to preserve RNA -tissue from organ shaved off to ~.1g to begin RNA isolation kit (2 organs, 6 snakes, 12 total samples)
may not have sampled right part of organ, may have sampled connective tissue rather than proper organ
-use mRNA to make cDNA copy, cDNA more stable ---used magnetic beads with TTTTTT (poly T) end that binds to AAAAA (poly A), use magnet to attract beads, attached are mRNA ---High throughput sequencing gives only short reads (~75 base pairs from each end) -fragment mRNA into smaller strands so that with short reads ^^ each bp can be read -use reverse transcriptase (RT)- enzyme in HIV/retroviruses- to form cDNA from mRNA + nucleotides -primers made of every possible 6 letter bp combination --> allows us to amplify every primer fragment
